Lenie Dijkshoorn

An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii

Since the 1970s, the spread of multidrug-resistant (MDR) Acinetobacter strains among critically ill, hospitalized patients, and subsequent epidemics, have become an increasing cause of concern. Reports of community-acquired Acinetobacter infections have also increased over the past decade. A recent manifestation of MDR Acinetobacter that has attracted public attention is its association with infections in severely injured soldiers. Here, we present an overview of the current knowledge of the genus Acinetobacter, with the emphasis on the clinically most important species, Acinetobacter baumannii.

The Population Structure of Acinetobacter baumannii: Expanding Multiresistant Clones from an Ancestral Susceptible Genetic Pool

Outbreaks of hospital infections caused by multidrug resistant Acinetobacter baumannii strains are of increasing concern worldwide. Although it has been reported that particular outbreak strains are geographically widespread, little is known about the diversity and phylogenetic relatedness of A. baumannii clonal groups. Sequencing of internal portions of seven housekeeping genes (total 2,976 nt) was performed in 154 A. baumannii strains covering the breadth of known diversity and including representatives of previously recognized international clones, and in 19 representatives of other Acinetobacter species. Restricted amounts of diversity and a star-like phylogeny reveal that A. baumannii is a genetically compact species that suffered a severe bottleneck in the recent past, possibly linked to a restricted ecological niche. A. baumannii is neatly demarcated from its closest relative (genomic species 13TU) and other Acinetobacter species. Multilocus sequence typing analysis demonstrated that the previously recognized international clones I to III correspond to three clonal complexes, each made of a central, predominant genotype and few single locus variants, a hallmark of recent clonal expansion. Whereas antimicrobial resistance was almost universal among isolates of these and a novel international clone (ST15), isolates of the other genotypes were mostly susceptible. This dichotomy indicates that antimicrobial resistance is a major selective advantage that drives the ongoing rapid clonal expansion of these highly problematic agents of nosocomial infections.

Standardization and Interlaboratory Reproducibility Assessment of Pulsed-Field Gel Electrophoresis-Generated Fingerprints of Acinetobacter baumannii

ABSTRACT A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.

Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13TU).

Acinetobacter genomic species (gen. sp.) 3 and gen. sp. 13TU are increasingly recognized as clinically important taxa within the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. To define the taxonomic position of these genomic species, we investigated 80 strains representing the known diversity of the ACB complex. All strains were characterized by AFLP analysis, amplified rDNA restriction analysis and nutritional or physiological testing, while selected strains were studied by 16S rRNA and rpoB gene sequence analysis, multilocus sequence analysis and whole-genome comparison. Results supported the genomic distinctness and monophyly of the individual species of the ACB complex. Despite the high phenotypic similarity among these species, some degree of differentiation between them could be made on the basis of growth at different temperatures and of assimilation of malonate, l-tartrate levulinate or citraconate. Considering the medical relevance of gen. sp. 3 and gen. sp. 13TU, we propose the formal names Acinetobacter pittii sp. nov. and Acinetobacter nosocomialis sp. nov. for these taxa, respectively. The type strain of A. pittii sp. nov. is LMG 1035(T) (=CIP 70.29(T)) and that of A. nosocomialis sp. nov. is LMG 10619(T) (=CCM 7791(T)).

Discrimination of Acinetobacter genomic species by AFLP fingerprinting.

AFLP is a novel genomic fingerprinting method based on the selective PCR amplification of restriction fragments. The usability of this method for the differentiation of genomic species in the genus Acinetobacter was investigated. A total of 151 classified strains (representing 18 genomic species, including type, reference, and field strains) and 8 unclassified strains were analyzed. By using a single set of restriction enzymes (HindIII and TaqI) and one particular set of selective PCR primers, all strains could be allocated to the correct genomic species and all groups were properly separated, with minimal intraspecific similarity levels ranging from 29 to 74%. Strains belonging to genomic species 8 (Acinetobacter lwoffii sensu stricto) and 9 grouped together in one cluster. The closely related DNA groups 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3 and 13TU (sensu Tjernberg & Ursing 1989) were clearly distinguishable, with intraspecific linkage levels above 50%. Strains of the independently described genomic species 13BJ (sensu Bouvet & Jeanjean 1989) and 14TU linked together at a relatively low level (33%). Although a previous DNA-DNA hybridization study seemed to justify the unification of these genomic species, AFLP analysis actually divides the 13BJ-14TU group into three well-separated subgroups. Finally, four unclassified strains obtained from diverse sources and origins grouped convincingly together, with a similarity linkage level of approximately 50%. These strains showed no similarities in their AFLP patterns with any of the other 155 strains studied and may represent a thus-far-undescribed Acinetobacter species. Based on these results, AFLP should be regarded as an important auxiliary method for the delineation of genomic species. Furthermore, because AFLP provides a detailed insight into the infraspecific structure of Acinetobacter taxa, the method also represents a highly effective means for the confirmation of strain identity in the epidemiology of acinetobacters.

Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp. nov., isolated from human clinical specimens

The taxonomic status of two recently described phenetically distinctive groups within the genus Acinetobacter, designated phenon 1 and phenon 2, was investigated further. The study collection included 51 strains, mainly of clinical origin, from different European countries with properties of either phenon 1 (29 strains) or phenon 2 (22 strains). DNA-DNA hybridization studies and DNA polymorphism analysis by AFLP revealed that these phenons represented two new genomic species. Furthermore, 16S rRNA gene sequence analysis of three representatives of each phenon showed that they formed two distinct lineages within the genus Acinetobacter. The two phenons could be distinguished from each other and from all hitherto-described Acinetobacter (genomic) species by specific phenotypic features and amplified rDNA restriction analysis patterns. The names Acinetobacter ursingii sp. nov. (type strain LUH 3792T = NIPH 137T = LMG 19575T = CNCTC 6735T) and Acinetobacter schindleri sp. nov. (type strain LUH 5832T = NIPH 1034T = LMG 19576T = CNCTC 6736T) are proposed for phenon 1 and phenon 2, respectively. Clinical and epidemiological data indicate that A. ursingii has the capacity to cause bloodstream infections in hospitalized patients.

Naturally transformable Acinetobacter sp. strain ADP1 belongs to the newly described species Acinetobacter baylyi.

ABSTRACT Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable Acinetobacter sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species Acinetobacter baylyi. Transformation assays demonstrate that the A. baylyi type strain B2T and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus Acinetobacter, characterized by the high transformability of its strains that have been cultured thus far.

Emergence of carbapenem resistance in Acinetobacter baumannii in the Czech Republic is associated with the spread of multidrug-resistant strains of European clone II

OBJECTIVES The aim of this study was to analyse the emergence of carbapenem resistance among hospital strains of Acinetobacter in the Czech Republic. METHODS Acinetobacter isolates were collected prospectively in 2005-06 from 19 diagnostic laboratories. They were identified to species level by AFLP, typed using AFLP, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing, and tested for susceptibility to 14 antimicrobials and for the presence of 20 genes associated with antimicrobial resistance. RESULTS A total of 150 Acinetobacter isolates were obtained from 56 intensive care units of 20 hospitals in 15 cities. They were identified as Acinetobacter baumannii (n = 108) or other species. A. baumannii isolates were allocated to EU clone I (n = 5), EU clone II (n = 66) or other, mostly unique genotypes. Two-thirds of the clone II isolates had nearly identical AFLP and PFGE fingerprints. As many as 85% and 88% isolates were susceptible to meropenem and imipenem (<or=4 mg/L), respectively. Carbapenem MICs of >or=8 mg/L were found in 23 A. baumannii isolates, of which 20 belonged to clone II. Isolates with bla(OXA-58-like) (n = 3)(,) bla(OXA-24-like) (n = 1) or ISAba1 adjacent to bla(OXA-51-like) (n = 34) had carbapenem MICs of 2 to >16 mg/L, while those without these elements showed MICs of <or=0.5-4 mg/L. Clone II isolates varied in susceptibility to some antibiotics including carbapenems and carried 6-12 resistance genes in 17 combinations. CONCLUSIONS The emergence of Acinetobacter carbapenem resistance in the Czech Republic is associated with the spread of A. baumannii strains of EU clone II. The variation in susceptibility in these strains is likely to result from both the horizontal spread of resistance genes and differential expression of intrinsic genes.

Horizontal Gene Transfer in a Polyclonal Outbreak of Carbapenem-Resistant Acinetobacter baumannii

ABSTRACT In the last few years, phenotypically carbapenem resistant Acinetobacter strains have been identified throughout the world, including in many of the hospitals and intensive care units (ICUs) of Australia. Genotyping of Australian ICU outbreak-associated isolates by pulsed-field gel electrophoresis of whole genomic DNA indicated that different strains were cocirculating within one hospital. The carbapenem-resistant phenotype of these and other Australian isolates was found to be due to carbapenem-hydrolyzing activity associated with the presence of the blaOXA-23 gene. In all resistant strains examined, the blaOXA-23 gene was adjacent to the insertion sequence ISAba1 in a structure that has been found in Acinetobacter baumannii strains of a similar phenotype from around the world; blaOXA-51-like genes were also found in all A. baumannii strains but were not consistently associated with ISAba1, which is believed to provide the promoter required for expression of linked antibiotic resistance genes. Most isolates were also found to contain additional antibiotic resistance genes within the cassette arrays of class 1 integrons. The same cassette arrays, in addition to the ISAba1-blaOXA-23 structure, were found within unrelated strains, but no common plasmid carrying these accessory genetic elements could be identified. It therefore appears that antibiotic resistance genes are readily exchanged between cocirculating strains in epidemics of phenotypically indistinguishable organisms. Epidemiological investigation of major outbreaks should include whole-genome typing as well as analysis of potentially transmissible resistance genes and their vehicles.

Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping.

ABSTRACT Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A−/B+) strains. Thirty-nine clinical toxin A−/B+ isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods. Furthermore, the toxin profile and clindamycin resistance were determined. Reference strains of C. difficile representing 30 known serogroups were also included in the analysis. AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types. The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar. Typing of 39 toxin A−/B+ isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes. Of 39 toxin A−/B+ isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%). A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A. Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A−/B+ isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test). We conclude that clindamycin-resistant C. difficile toxin A−/B+ strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.